Nucleoside analogues

ABSTRACT

Compositions for topical use in herpes virus infections comprising anti-herpes nucleoside analogue phosphate esters, such as acyclovir monophosphate and acyclovir diphosphate, which show increased activity against native strains of herpes virus as well as against resistant strains, particularly thymidine kinase negative strains of virus. Also disclosed are methods for using the topical compositions in treatment of herpes disease.

RELATED APPLICATIONS

This application is a continuation in part of U.S. patent applicationSer. No. 07/777,683, filed Oct. 15, 1991, now abandoned, the disclosureof which is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the topical treatment of cutaneousvirus infections. It relates particularly to the topical treatment ofherpes simplex infections, including herpes simplex types 1 and 2, usingformulations comprising phosphate esters of anti-herpes antiviralnucleoside analogues such as acyclovir.

2. Background of the Art

Acyclovir (ACV) is an antiviral nucleoside analogue of guanosine whichcontains an unusual incomplete (acyclic) sugar moiety. Nucleosideanalogues interrupt the process of DNA replication in cells, and are forthat reason useful as antiviral and antineoplastic agents. ACV isparticularly effective in treating herpes simplex virus infections oftypes I and II. The antiherpes virus activity of ACV in cells occurswith low toxicity because ACV is selectively phosphorylated by HSVthymidine kinase, but not host cell thymidine kinase. As a consequence,only cells infected with HSV can form ACV monophosphate (ACV-MP). ACV-MPis then anabolically converted by cellular enzymes to ACV triphosphate,the active agent that interferes with viral replication. (Fyfe, J., etal., J. Biol. Chem. 253:8721-8727 (1978); Furman, P., et al., J. Virol.32:72-77 (1979)).

The anti-herpes virus activity of acyclovir has been demonstrated ininhibiting the replication of herpes simplex virus in tissue culturecells (O'Brien, W., et al., Antimicrob. Agents and Chemother.34:1178-1182 (1990); it has also been demonstrated in clinical studieswherein patients infected with HSV were treated with orally administeredACV (Whitley, R., Immunobiol. and Prophylaxis of Human HerpesvirusInfections, C. Lopez et al, (eds) Plenum Press, NY 1990; and Straus, S.,Sexually Transmitted Diseases 16(2):107-113 (1989).

Acyclovir is the treatment of choice for mucosal and cutaneous HSVinfections, although, patients receiving topical acyclovir therapyexperience some reductions of their symptoms, healing is slow andincomplete (Spruance, S., et al., J. Infect. Dis. 146:85-90 (1982); andSpruance, S., et al., Antimicrob. Agents Chemother. 25:553-555 (1984).

Combination treatment using ACV together with interferon for herpesvirus infected cultured cells (O'Brien, W., et al., Antimicrob. Agentsand Chemother. 34(6):1178-1182 (1990) or using ACV together with A1110U,an HSV inactivator, as a topical therapy for herpetic keratitis inathymic mice (Lobe, D., et al., Antiviral Research 15:87-100 (1991)showed synergistic antiherpesvirus I activity over the use of ACV alone.

Acyclovir has been used with qualified success in a clinical trial totreat another viral disease, varicella (chickenpox) (Whitley, R., etal., Immunobiology and Prophylaxis of Human Herpesvirus Infections, C.Lopez (ed), Plenum Press, New York (1990) pp. 243-253. It has also beenused experimentally but without success in treating other disorders inwhich a viral etiology was hypothesized, such as aplastic anemia(Gomez-Almaguer, D., et al. Amer. J. of Hematology 29:172-173 (1988) andduodenal ulcer (Rune, S. J., et al., Gut 31:151-152 (1990)).

Acyclovir phosphates have been shown to be efficacious against wild typeor laboratory isolates of HSV-1 infected cultured cells in vitro, buthave little or no efficacy against thymidine kinase defective mutants ofHSV under the same conditions. (See data of FIGS. 1 and 2).

In immunosuppressed patients, such as those with HIV (AIDS) infectionsor transplant recipients who are taking immunosuppressive drugs toprevent transplant rejection, ACV has been given chronically to preventtroublesome outbreaks of herpes. Such therapy provides a selectivepressure which leads to mutations in HSV thymidine kinase (90%frequency) as well as DNA polymerase (10% frequency), which in turnresults in ACV-resistant viral strains. There is no effective topicaltherapy for these acyclovir resistant herpes virus strains.

SUMMARY OF THE INVENTION

According to the invention, there are provided acyclovir phosphateesters and other antiherpes antiviral nucleoside analogue phosphateesters that are effective in the treatment of mucosal and cutaneousherpetic lesions due to herpes virus infections. These agentssurprisingly show antiviral activity against lesions due to thymidinekinase defective herpes virus infections, even though they arerelatively inactive against these mutant viruses in cultured cells. Theinvention also provides pharmaceutical formulations, comprising aneffective, antiviral concentration of an acyclovir derivative which canbe acyclovir monophosphate, acyclovir diphosphate, acyclovirmonophosphate glycerol, acyclovir diphosphate glycerol, acyclovirmonophosphate morpholidate, acyclovir monophosphate glycerol, acyclovirdiphosphate glycerol, acyclovir monophosphate isopropylidene glycerol,acyclovir diphosphate isopropylidene glycerol, or a mixture thereof, ina pharmaceutical carrier suitable for topical use.

Other antiherpes simplex nucleosides which rely on phosphorylation byviral thymidine kinase will also exhibit enhanced activity when appliedto the skin of infected patients as their phosphate esters in a suitabletopical formulation.

According to another aspect of the invention, there is provided a methodfor the topical treatment of a viral infection, comprising applying aformulation comprising any of the acyclovir phosphate derivatives of theinvention, or a mixture thereof, to the mucosal or cutaneous lesions ofa virus infected animal, including a human or other mammal. In apreferred embodiment of the method, the animal is infected with a herpesvirus. In a particularly preferred embodiment, the animal is infectedwith a herpes virus strain that is resistant to acyclovir. The acyclovirresistant herpes virus strain can be a viral strain in which resistanceto the antiviral agent is due to an alteration or defect in thethymidine kinase gene.

In accordance with another aspect of the present invention, ananti-herpes nucleoside analogue monophosphate is used in the preparationof a medicament for the treatment of a mucosal or cutaneous viralinfection. In a preferred embodiment, the nucleoside monophosphate is awater soluble salt. In another preferred embodiment, the viral infectionis herpes simplex virus, type 1 or type 2.

In another aspect of the present invention, the anti-herpes nucleosideanalogue monophosphate is used together with a pharmaceuticallyacceptable carrier in the preparation of a medicament for the treatmentof a mucosal or cutaneous viral infection. In a preferred respect, thepharmaceutically acceptable carrier is selected from the groupconsisting of an aqueous cream and polyethylene glycol.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the comparative effect of acyclovir and acyclovirmonophosphate on herpes simplex virus replication in Wi38 fibroblasts.

FIG. 2 illustrates the comparative effect of acyclovir and acyclovirmonophosphate on the replication of HSV-DM.21 TK mutant in vitro.

FIG. 3 illustrates the comparative effect of topical acyclovir andacyclovir phosphate esters on acyclovir-resistant HSV-1 infections ofthe TK-deficient type in HRS/J mice.

FIG. 4 illustrates the comparative effect of topical acyclovir andacyclovir phosphate esters on acyclovir-resistant HSV-1 infections ofthe TK-altered type in HRS/J mice.

FIG. 5 illustrates the comparative effect of topical acyclovir andacyclovir monophosphate on acyclovir-resistant HSV-1 infections of thewild type in HRS/J mice.

FIG. 6 illustrates the comparative effect of topical acyclovir andacyclovir monophosphate on acyclovir-resistant HSV-1 infections of theTK-altered type in HRS/J mice.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides acyclovir phosphate derivatives thatdemonstrate excellent topical activity against herpes simplex virus(HSV) infections, particularly against ACV-resistant mutants of HSV.

Acyclovir is an analogue of the purine base, guanine, having asubstituent group at the 9-position, and having an acyclic sugar groupfrom which the common name is derived. The chemical name of acyclovir is9-(2-hydroxyethoxymethyl)guanine, which has the structure: ##STR1##

The acyclovir phosphate derivatives of the invention have a substituent,R, at the terminal O-locant of the acyclic sugar group, as follows:##STR2## wherein the R substituents are as follows: ##STR3##

According to the invention, acyclovir monophosphate (ACV-MP), acyclovirdiphosphate (ACV-DP), acyclovir monophosphate glycerol (ACV-MP-G),acyclovir diphosphate glycerol (ACV-DP-G), acyclovir monophosphatemorpholidate (ACV-MP-morpholine), acyclovir monophosphate isopropylideneglycerol (ACV-MP-isoP-G), or acyclovir diphosphate isopropylideneglycerol (ACV-DP-isoP-G), either alone or combined, are prepared in asuitable topical pharmaceutical formulation and applied to the cutaneouslesions of an HSV-infected individual. The compounds ACV-MP, ACV-DP,ACV-DP-G, the morpholine derivative of acyclovir, and the acyclovirisopropylidene glycerol derivatives described, are non-lipid, watersoluble phosphate esters, and are therefore preferably provided in anaqueous base topical formulation.

Surprisingly, we have discovered that the monophosphates of acyclovir,and we expect that monophosphate derivatives of other nucleosides will,exhibit enhanced topical anti-HSV activity. We have also demonstratedthat salts of monophosphate derivatives nucleosides can be easilyprepared, and that such salts exhibit enhanced solubility in aqueousmedia, i.e., cream, gels, or other aqueous dispersions. Moreover, suchsalts are soluble in polyethylene glycol media which provides a uniquemucosal or cutaneous dispersion.

Similarly, monophosphates, diphosphates, and other phosphate esters ofother antiherpes simplex nucleosides will exhibit enhanced topicalactivity as those above noted. The following herpes. antiviralnucleosides exhibit enhanced activity as phosphate esters:

1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil

[broavir; BV-araU];

2'-fluorocarbocyclic-2'-deoxyguanosine;

6'-fluorocarbocyclic-2'-deoxyguanosine;

1-(beta-D-arabinofuranosyl)-5(E)-(2-iodovinyl)uracil;

SQ 34,514;

HOE 602;

triflurothymidine;

9-[(1,3-dihydroxy-2-propoxy)methyl]guanine;

5-ethyl-2'-deoxyuridine;

E-5-(2-bromovinyl)-2'-deoxyuridine;

5-(2-chloroethyl)-2'-deoxyuridine;

1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine (FIAC);

1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouridine (FIAU);

buciclovir;

6-deoxyacyclovir;

9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine;

E-5-(2-iodovinyl)-2'-deoxyuridine;

5-vinyl-1-beta-D-arabinofuranosyluracil (V-araU);

1-beta-D-arabinofuranosulthymine (Ara-T);

2'-nor-2'deoxyguanosine (2'NDG);

9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine (penciclovir, BRL 3912)

1-beta-D-arabinofuranosyladenine (Ara-A; vidarabine)

The various phosphate esters of these compounds may be preparedessentially as described below for acyclovir.

Synthesis of Acyclovir Phosphate Esters

The present invention provides methods for the synthesis of acyclovirmono- and diphosphates, acyclovir monophosphate morpholidates, acyclovirmono- and diphosphate glycerols, and acyclovir mono- and diphosphate1,2-isopropylidene glycerol.

Acyclovir monophosphate can be prepared from acyclovir according to themethod of Yoshikawa, M., et al., Bull. Chem. Soc. Japan 42:3505-3508(1969) as modified by the method of Toorchen, D. and Topal, M.,Carcinogenesis 4:1591-1597 (1983), and exemplified in Example 1.Acyclovir diphosphate can be prepared, in the manner of other nucleosideanalogues, by the method of Ott, D. G., et al., Anal. Biochem.21:469-472 (1967), using either tributylammonium phosphate ortributylammonium pyrophosphate as the phosphate donor.

Methods for the preparation of acyclovir diphosphate glycerol arepresented in Examples 2 through 4. In general, the nucleoside phosphateglycerols are prepared in a manner similar to that for the preparationof phosphatidyl nucleosides. In the approach described in Example 3,acyclovir phosphate is activated by the addition of a leaving group, forexample, morpholine, according to Example 2, and condensed withglycerol-3-phosphate dicyclohexylammonium salt in the presence ofN,N'-dicyclohexylcarbodiimide (DCC). Alternatively, as described inExample 4, glycerol phosphate, having the reactive hydroxyl groupsprotected by an isopropylidene moiety, is activated by addition ofmorpholidate, and then condensed with acyclovir monophosphate under theconditions described for Example 2.

A number of acyclovir-diphosphate-diglycerides (ACV-DP-DG) containingvarious acyl chains have been prepared in the past by the condensationof the appropriate diacylphosphatidic acid morpholidate(PA-Morpholidate) and acyclovir monophosphate (ACV-MP.) A method bywhich the procedure can be carried out is described by Agranoff, B. andSuomi, W., Biochem. Prep. 10:47-51 (1963). Alternatively, themorpholidate of the nucleoside monophosphate is prepared and condensedwith a phosphatidic acid as described in U.S. patent application Ser.No. 07/706,873 entitled "Liponucleotide Synthesis," and by Hong, et al.,British Patent Application No. 2,168,350.

The chemical methods above are generally disclosed in terms of theirgeneral application to the preparation of compounds of the invention.Occasionally, the reaction may not be applicable as described to eachcompound included within the disclosed scope. The compounds for whichthis occurs will be readily recognized by those skilled in the art. Inall such cases, either the reactions can be successfully performed byconventional modifications known to those skilled in the art, e.g. byappropriate protection of interfering groups, by changing to alternativeconventional reagents, or by routine modification of reactionconditions. Alternatively, other reactions disclosed herein or otherwiseconventional will be applicable to the preparation of the correspondingcompounds of the invention. In all preparative methods, all startingmaterials are known or readily preparable from known starting materials.Unless otherwise indicated, all parts and percentages are by weight.

The acyclovir derivatives of the invention, comprising ACV-MP, ACV-DP,ACV-MP-glycerol, ACV-DP-glycerol, ACV-MP-isopropylidene glycerol, andACV-DP isopropylidene glycerol were found to have particular efficacy intopically treating the herpetic lesions of acyclovir-resistant HSV-1infections. Example 7 demonstrates that infection of cultured cells withwild type isolates and laboratory strains of HSV can be treated withequal success using acyclovir, acyclovir monophosphate (Example 7; FIG.1). For these viral infections in Wi38 fibroblasts, both acyclovir andacyclovir monophosphate have the same IC₅₀ of about 1 or 2 μMconcentration. However, when the same cultured cell system is infectedwith an acyclovir-resistant strain of virus, HSV-DM.21, lacking thethymidine kinase necessary to convert acyclovir to acyclovirmonophosphate, acyclovir and acyclovir monophosphate are ineffective inreducing the number of viral plaques (Example 7; FIG. 2).

The efficacy of acyclovir phosphate esters with respect toacyclovir-resistant cutaneous HSV-1 infections is surprising in view ofthe cultured cell in vitro data above. Acyclovir phosphate estersapplied in an aqueous cream to the herpetic lesions of mice infectedwith acyclovir-resistant HSV-1 were substantially more effective thannative acyclovir in reducing the number of such lesions (Example 9;FIGS. 3 and 4).

Accordingly, in view of these results, it is believed that in vitroincorporation of acyclovir, and acyclovir phosphates, proceed through adifferent mode of operation than in vitro as a topically applied lotion.

Topical Formulations of Nucleoside Knalogue Phosphates

The nucleoside analogue derivatives of the invention as previouslydescribed can be prepared for topical use by incorporation into avariety of formulations known to those in the art as useful andconvenient for dermatological use. The nucleoside analogue derivativesare water soluble, and accordingly an aqueous solution, water-in-oilemulsion, or an aqueous cream are highly preferred formulations. Watersolubility of the acyclovir and other nucleoside monophosphates can beenhanced through the preparation of salts, such as sodium, potassium,ammonium, or hydrogen. In a particularly preferred formulation, theactive ingredient is prepared in a polyethylene glycol (PEG) vehicle.Alternatively, the active ingredients can be topically applied in a drypowder formulation, using an insoluble powder, such as starch or talc asa diluent or carrier.

The vehicle is an important component of some topical formulations,because it can be selected to enhance penetration, to prolong theduration of activity, or to meet requirement of the site of application.For example, a formulation for application to the callous parts of thebody, such as the palms of the hand or bottoms of the feet, can includea penetration enhancing agent such as dimethylsulfoxide propylene glycolor azone™; a powdery formulation can be selected for application to theintertriginous zones such as the crotch, inner elbow or between thefingers or toes. The formulation can also be made up to contain variousorganic polymers or other compositions known to those of skill in theart to give sustained release of the active antiviral acyclovirderivatives.

A multitude of appropriate topical formulations can be found in theformulary known to all pharmaceutical chemists: Remington'sPharmaceutical Sciences, 15th Edition, 1975. Mack Publishing Company,Easton, Pa. 18042. (Chapter 87: Blaug, Seymour). These formulationsinclude for example, powders, pastes, ointments, jelly, waxes, oils,lipids, anhydrous absorption bases, oil-in-water or water-in-oilemulsions, emulsions carbowax (polyethylene glycols of a variety ofmolecular weights), semi-solid gels, and semisolid mixtures containingcarbowax.

The concentration of active ingredient in the topical formulations ofthe invention can be from about 0.01 gm% to 100 gm%; preferably fromabout 0.1 gm% to 50 gm%; most preferably from about 1 gm% to about 15gm%. The formulations can further comprise effective concentrations ofother agents which help to promote penetration of the skin and healing,as described in the above-referenced formulary and are well known tothose of ordinary skill in the art.

Efficacy of topical formulations containing the active phosphate estersof the invention can be evaluated using conventional testing procedures,known to those of skill in the art. For example, a particularlyexpeditious procedure is the murine "orofacial model," as described byEllis, M., et al., Antimicrobial Agents and Chemotherapy 33:304-310(1989). In this test system, the pathogenesis of HSV in mice scarifiedand inoculated on the snout has been shown to be a reasonable model ofthe disease cycle of cutaneous HSV infection in the immunocompromisedhost.

The formulations can be applied to the herpetic lesions of the affectedskin repeatedly; for example once, twice, or several times a day, andthe treatment can be extended for several days until healing isachieved. The risk of incidence of toxicity and irritation is minimal.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the invention to itsfullest extent.

The present invention is described below in additional detail using thefollowing examples, but the methods described below are applicable toall methods within the scope of the invention and the invention is notlimited to the examples given. The following preferred embodiments are,therefore, to be construed as merely illustrative and not limitative ofthe remainder of the disclosure in any way whatsoever.

EXAMPLE I Phosphorylation Of Acyclovir Nucleoside Derivatives

We prepared acyclovir nucleoside derivatives through the followingmethods. Unprotected acyclovir was reacted with POCl₃ in trimethylphosphate ((CH₃ O)₃ PO) was performed essentially as described byYoshikawa et al. Tetrahedron Letters 50:5065-5068 (1967); and Yoshikawa,M., Kato et al. Bull Chem. Soc. Japan 42:3205-3208 (1967). To a cooledsolution (0° C.) of 2M POCl₃ in 300-400 ml trimethyl phosphate,acyclovir (1M) was added dropwise with stirring, the reactiontemperature being held constant between 0° and 5° C. The progress of thereaction was monitored by means of HPLC using a Mono Q HR 5/5 anionexchange column (Pharmacia, Uppsala, Sweden). Typically, 5 μl of thereaction mixture was neutralized with aqueous sodium hydroxide (final pH7) and injected on the column.

Elution was performed as follows: washing with water, elution with 0.1Mammonium carbonate, NH₄ HCO₃, which elutes the acyclovir monophosphate,followed by a linear gradient of 0.1-0.6M NH₄ HCO₃ which elutes somehigher phosphorylated products. The reaction was mostly completed within45 to 75 minutes as determined by this method, and the reaction productwas hydrolyzed and neutralized with 2 volumes of aqueous sodiumhydroxide to final pH of 7.

Purification of the product compound was conducted as described abovefor the analysis of the reaction mixture. By this method, 0.8 moles ofacyclovir monophosphate were prepared and purified with a Q Sepharosefast flow column using the same elution conditions.

Yields varied between 80% and 96% after repeated lyophilization fromwater.

TLC analysis (Silica 60/F254 Plates, Merck) showed a single U.V. and Pipositive spot, using the developing system 1-propanol/25% NH₃ /H² O(20:20:3 by volume)

EXAMPLE II Preparation Of Acyclovir-monophosphomorpholidate

Acyclovir-monophosphate (5 mmol) and morpholine (20 mmol) were suspendedin t-butanol (50 ml) and heated under gentle reflux whileN,N'-dicyclohexylcarbodiimide (DCC, 20 mmol) dissolved in t-butanol (50mmol) was added dropwise over a period of 1 hour. The mixture wasstirred under reflux for 12 to 36 hours and evaporated to dryness. Theresidue was triturated with ether and washed by decantation with thesame solvent. The product was purified by recrystallization frommethanol-ether mixtures.

EXAMPLE III Preparation Of sn-Glycero-3-diphospho-acyclovir FromAcyclovir-monophosphomorpholidate

Acyclovir monophosphomorpholidate (2 mmol), prepared as described inExample 2, was dissolved in dry pyridine (20 ml) and evaporated todryness under vacuum. The process of dissolving the residue in pyridineand evaporation was repeated three additional times to remove traces ofwater from the compound. Glycerol-3-phosphate dimonocyclohexylammmoniumsalt (3 mmol) was added to the dried residue and the mixture wasdissolved in 20 ml of pyridine and stirred under inert atmosphere at 60°C. for 12-36 hours. The solvent was evaporated under vacuum and theresidue was titrated with ether and the resulting solid was purified byion exchange chromatography over DEAE sephadex column (2.5 cm ×30 cm)using a linear gradient of ammonium bicarbonate (10 mmol to 300 mmol,500 ml each). Fractions containing pure product (identified by using TLCand analytical HPLC) were pooled and lyophilized to furnish the titlecompound.

EXAMPLE IV Preparation Of sn-Glycero-3-diphospho-acyclovir From1,2-isopropylidene-sn-glycero-3-monophosphomorpholidate A. PreparationOf 1,2-Isopropylidene-sn-glycero-3-phosphate

Phosphorous oxychloride (25 mmol) was added dropwise over a period of 30minutes to a mixture of 1,2-Isopropylidene-glycerol (20 mmol) (Sigma,St. Louis, Mo.) and triethylamine (100 mmol) that was cooled to 0° C.After stirring the mixture at 0° C. for 10 to 90 minutes. Water (1 ml)was added to stop the reaction. The mixture was then dissolvedchloroform (500 ml) and washed with water (3×100 ml). The water washsolutions were combined and back extracted with chloroform (50 ml) andlyophilized. The product was used immediately for subsequent reactionswithout any additional purification.

B. Preparation Of1,2-isopropylidene-sn-glycero-3-monophosphomorpholidate

1,2-isopropylidene-sn-glycero-3-phosphate, prepared as described in (A),was condensed with morpholine to prepare1,2-isopropylidene-sn-glycero-3-monophosphomorpholidate, according tothe procedure described for the preparation of acyclovirphosphomorpholidate in Example 2.

Reaction of the intermediate compound,1,2-isopropylidene-sn-glycero-3-monophosphomorpholidate, with acyclovirmonophosphate under the conditions described in Example 2, yielded1,2-isopropylidene-sn-glycero-3-monophosphomorpholidate.

1,2-isopropylidene-sn-glycero-3-diphospho-acyclovir (1 mmol) wasdissolved in 50 to 90% aqueous acetic acid and stirred at roomtemperature for a period of 4 to 12 hours and the crudeglycero-3-diphospho-acyclovir product was purified as described above.

EXAMPLE V Preparation Of sn-glycero-3-phospho-acyclovir

1,2-Isopropylidene-sn-glycero-3-phosphate, 1 mM (prepared as in Example4B), and acyclovir (1 mM), were suspended in dry pyridine (10 ml) andDCC (4 mmol). Dissolved pyridine (4 ml) was added and the mixturestirred at 25° C. to 60° C. for 12 to 72 hours. The solvent wasevaporated and the residue was titrated with ether. The crude productwas purified by ion exchange chromatography as described in Example 3.The isopropylidene-protecting group was then removed from the product bytreating with aqueous acetic acid to furnish the title compound.

Alternatively, the title compound was also prepared by using2,4,6-triisopropylbenzenesulfonyl chloride (TPS-Cl) as the condensingagent.

EXAMPLE VI Preparation Of Acyclovir Phosphate Ester Mixture By TheAlkaline Hydrolysis Of Acyclovir Diphosphate Dipalmitoylglycerol

Acyclovir-diphosphate-dipalmitoylglycerol (1 mmol) was dissolved inchloroform, to which methanolic sodium hydroxide (2.1 mmol) was added.The reaction was carried for 20 to 90 minutes and the progress wasmonitored by TLC. Upon completion of the reaction, Dowex-50 X-2 (H+) wasadded to the reaction mixture to adjust the pH to 7. The resin wasseparated by filtration and the filtrate was lyophilized and the crudeproduct was purified as described in Example 1.

EXAMPLE VII Absence Of Antiviral Effect Of Acyclovir Monophosphate InAcyclovir-Resistant TK Mutant Strains Of HSV (DM.21)

Separate cultures of Wi-38 fibroblast cells infected with either wildtype strains of herpes simplex virus (HSV) or a mutant strain of HSV(DM.21) were individually treated with acyclovir, or acyclovirmonophosphate. The DM.21 mutant lacks the thymidine kinase enzyme whichusually converts ACV to ACV-MP, and is therefore resistant to acyclovir.The results for HSV-1 are shown in FIG. 1, and those for HSV-DM.21 areshown in FIG. 2. An IC₅₀ is that concentration of antiviral agent whichinhibits viral plaque formation 50%.

In wild type isolates and laboratory strains of herpes simplex virus(HSV-1), acyclovir and acyclovir monophosphate have IC₅₀ s of 0.1 μM(FIG. 1). In contrast, both acyclovir and acyclovir monophosphate haveIC₅₀ s in excess of 100 μM against mutant HSV strains in this assay(FIG. 2).

Based on these results in vitro, one would not expect acyclovirmonophosphate to exhibit significant activity when administeredtopically to animals infected with a thymidine kinase defective or othermutant strain of HSV.

EXAMPLE VIII Antiviral Effect Of Acyclovir Phosphate Esters In MiceInfected With Acyclovir-Resistant Strains Of HSV

Mice of the HRS/J type were infected cutaneously using the snoutscarification method as described by Ellis, M. et al., AntimicrobialAgents and Chemotherapy 33(3):304-310 (1989). Briefly, groups of 10mice, under light ether anesthesia, were inoculated on the snout byscarification with a 25-gauge needle, followed by 10 seconds of rubbingwith a cotton-tipped applicator soaked in diluted virus. The virus usedfor infection was a TK-deficient strain, referred to in Ellis, M. et al.(TK^(D)). Three hours post-infection, the animals were treatedtopically, 3 times daily, for 4 days, with formulations of acyclovir oracyclovir phosphates, in a aqueous cream (AC), according to the Ellisreference cited above.

The results are presented in FIG. 3. A formulation comprising 5 gm%acyclovir was active. In contrast, a formulation comprising 5 gm% of amixture of 80% acyclovir monophosphate together with 20% other acyclovirphosphate esters (acyclovir diphosphate and acyclovir diphosphateglycerol) showed superior activity, with only a few mice developingherpetic lesions. All lesions were healed by day 8 in all groups.

The above procedure was repeated, with treatment continuing for 5 days,using the TK-altered HSV-1 virus (TK^(A), Ellis, above), a more virulentstrain. Unlike the TK^(D) virus, TK^(A) is fatal in untreated mice.Treatment with 5 gm percent acyclovir reduced lesion scores moderately,with most animals surviving and improving substantially by day 14. Withthe same concentration of phosphate esters, however, there was adramatic improvement in lesion scores, with all lesions resolved after 9days, and all animals surviving, as shown in FIG. 4.

EXAMPLE IX Antiviral Effect Of Acyclovir Monophosphate In Mice InfectedWith An Acyclovir Resistant Wild Type HSV-1

The procedure of Example 8 was repeated using an acyclovir-sensitive,wild type HSV-1 and a formulation having only acyclovir monophosphate(ACV-MP) as the acyclovir derivative. Two creams were formulated, onehaving ACV-MP present in the aqueous cream at 14.5 millimoles/100 ml andthe other having acyclovir present at 22.2 millimoles/100 ml (both 5gm%, however, because of the addition of the phosphate group the numberof moles of acyclovir present in the monophosphate is reduced relativeto neat acyclovir).

Treatment was initiated 24 hours after infection and continued 4 timesdaily for four days. The ten untreated mice developed stage 4 lesions bythe 5th day and all died by day 14 (FIG. 5). The acyclovirmonophosphate-treated animals did not develop lesions and 10 of 10animals survived (FIG. 5). In the acyclovir-treated group severalanimals developed mild lesions on days 7-9 which resolved; 9 of 10 tenanimals survived.

This study shows that ACV-MP at a lower dosage (14.5 mmol/100 ml) wasmore effective than acyclovir (22.2 mmol/100 ml) in preventing lesionsin wild type HSV-1 infection.

EXAMPLE X Antiviral Effect Of Acyclovir Monophosphate In Mice InfectedWith An Acyclovir Resistant HSV-1

The procedure of Example 8 was repeated using a formulation having onlyacyclovir monophosphate (ACV-MP) as the acyclovir derivative. Treatmentwas begun 3 hours post-infection, with treatments occurring twice on theday of infection, and thereafter, three times a day through day 4.Referring now to FIG. 6, ACV-MP at 14.5 mmol/100 ml is clearly moreeffective than acyclovir at 22.2 mmol/100 ml in reducing lesion scoresin animals infected with acyclovir-resistant (TK altered) HSV-1.

In the control and acyclovir-treated groups, 8 of 10 mice survived the14 day experiment versus 10 of 10 surviving with acyclvoir monophosphatetreatment.

EXAMPLE XI Antiviral Effect Of Acyclovir Monophosphate

In Guinea Pigs Infected With An Acyclovir Resistant HSV-2

We tested acyclovir monophosphate (ACV-MP) in aqueous cream (AC) todetermine if it was more effective than 5% Acyclovir in polyethyleneglycol (ACV-PEG) for treatment of a primary genital herpes. Inparticular, we studied a genital herpes infection of guinea pigs causedby an ACV-resistant HSV-2. Additionally, we compared acyclovirtreatments in two carrier systems: aqueous cream (AC) and polyethyleneglycol (PEG). The experiments were placebo-controlled and uninfectedanimals were treated with each ACV preparation to assess skin andvaginal irritation.

Intravaginal inoculation of weanling guinea pigs with HSV-2 results in aprimary genital infection is characterized by initial replication ofvirus in the vaginal tract followed by the development of externalvesicular lesions. Virus titers peak on days one to three in the vaginaltract and gradually clear by days 7-10. The external genital lesionsfirst appear on day four, peak lesion severity occurs on days 6-8, andthe lesions generally heal by days 15-18.

Animals were inoculated with the HSV-2 strain 12247, which has analtered thymidine kinase and is resistant to in vitro treatment withACV. Female Hartley guinea pigs (Charles River, Kingston, N.Y.) weighing250-300 grams were first vaginally swabbed to remove vaginal secretions.After one hour, the animals were inoculated intravaginally with 2.4×10⁴plague forming units (pfu). Inoculation was accomplished by inserting aswab soaked with virus into the vaginal tract and rotating approximatelysix times.

Groups of 10 guinea pigs were treated both intravaginally and on theexternal genital skin with 0.1 ml (total of 0.2 ml per animal pertreatment) of each preparation. Animals were treated three times dailyfor seven days beginning 24 hours post-viral inoculation. Threeuninfected animals were treated with each preparation on the sameschedule to assess local toxicity and irritation.

To determine the efficacy of the various treatments on HSV-2 replicationin the vaginal tract, swabs of vaginal secretions were obtained duringthe primary infection on days 1, 3, 5, 7, and 10 after HSV-2inoculation. The swabs were placed in tubes containing 2.0 ml of media,vortexed, and frozen at -70° C. until titrated for HSV. When all sampleswere collected, they were thawed, diluted serially, and HSV-2 titerswere determined using rabbit kidney cells in a microtiter CPE assay. Wealso measured the development and severity of external genital lesionsto determine the efficacy of treatment. Severity of lesions was gradedon a 0-5+ score. The presence or absence and severity of lesions wasrecorded for 19 days after viral inoculation. Infection rates, peaklesion scores, peak virus titers, areas under virus titer-day curves,and lesion score-day curves between PBS placebo-treated and PEGdrug-treated or AC placebo-treated and AC drug-treated animals werecompared using the Mann-Whitney U rank sum test. A p-value of 0.05 orless was considered significant.

The effect of topical treatment with ACV preparations on vaginal viralreplication is shown in Table I. Only treatment with the ACV-MPpreparations (5% ACV-MP-PEG or 5% ACV-MP-AC) significantly reduced thevirus titer-day area under the curve (AUC) and mean peak virus titers.

                                      TABLE I                                     __________________________________________________________________________    EFFECT OF TREATMENT WITH ACYCLOVIR MONOPHOSPHATE ON                           VAGINAL VIRUS TITERS OF GUINEA PIGS INOCULATED                                INTRAVAGINALLY WITH AN ACYCLOVIR RESISTANT HSV-2                                      # Virus                                                                       Positive/#                                                                          Virus Titer-Day                                                                              Mean Peak                                        Treatment.sub.A                                                                       Inoculated                                                                          Area Under Curve                                                                        P-Value                                                                            Virus Titer                                                                         P-Value                                    __________________________________________________________________________    Placebo-PBS                                                                           10/10 31.6      --   5.0   --                                         Placebo-AC                                                                            10/10 34.6      NS.sup.B                                                                           5.1   NS                                         ACV-PEG 10/10 33.4      NS   5.2   NS                                         ACV-AC  10/10 27.9      NS   4.6   NS                                         ACV-MP-PEG                                                                             6/10 3.4       <0.001                                                                             2.0   <0.001                                     ACV-MP-AC                                                                              9/10 14.5      0.001                                                                              3.7   <0.05                                      __________________________________________________________________________     .sub.A. Topical and intravaginal treatment was initiated 24 hours after       viral inoculation and was continued three times daily for 7 days.             Acyclovir content on a molar basis was lower in the tests conducted with      acyclovir monophosphate (14.5 mmol/100 ml) versus those conducted with th     neat acyclovir (22.2 mmol/100 ml).                                            .sup.B. NS = Not Statistically Significant when compared to the               appropriate placebotreated group.                                        

The effect of topical treatment with ACV preparations on lesiondevelopment is depicted in Table II. Both ACV and ACV-MP preparationssignificantly altered the lesion score-day AUC when compared to theappropriate placebo-treated group. However, only therapy with 5%ACV-MP-PEG significantly reduced mean peak lesion scores.

                                      TABLE II                                    __________________________________________________________________________    EFFECT OF TREATMENT WITH ACYCLOVIR MONOPHOSPHATE                              ON EXTERNAL LESION DEVELOPMENT IN AN                                          ACYCLOVIR RESISTANT GENITAL HSV-2 INFECTION                                   OF GUINEA PIGS                                                                        Lesion Score-Day Area                                                                           Mean Peak                                           Treatment.sub.A                                                                       Under Curve  P-Value                                                                            Lesion Score                                                                          P-Value                                     __________________________________________________________________________    Placebo-PBS                                                                           28.3         --   3.0     --                                          Placebo-AC                                                                            34.2         NS.sub.B                                                                           3.5     NS                                          ACV-PEG 9.5          0.001                                                                              1.8     NS                                          ACV-AC  19.3         0.01 2.5     NS                                          ACV-MP-PEG                                                                            1.7          <0.001                                                                             0.7     <0.001                                      ACV-MP-AC                                                                             23.4         <0.05                                                                              2.3     NS                                          __________________________________________________________________________     .sub.A. Topical and intravaginal treatment was initiated 24 hours after       viral inoculation and was continued three times daily for 7 days.             Acyclovir content on a molar basis was lower in the tests conducted with      acyclovir monophosphate (14.5 mmol/100 ml) versus those conducted with th     neat acyclovir (22.2 mmol/100 ml).                                            .sub.B. NS = Not Statistically Significant when compared to the               appropriate placebotreated group.                                        

In the guinea pig model of an ACV-resistant HSV-2 genital herpesinfection, only ACV-MP significantly reduced vaginal viral replication.Also, the ACV-MP-PEG treated group had the lowest virus titer-day andmean peak titer values. While both ACV-MP and ACV altered lesiondevelopment, the drugs in PEG had lower scores than those in AC.Additionally, animals receiving ACV-MP-PEG had the lowest lesionscore-day and mean peak lesion scores.

Moreover, throughout the study, there were no signs of any irritation ofthe genital area or any other toxicity in uninfected ACVpreparation-treated animals.

These results demonstrate the strong activity of acyclovir monophosphatein treating HSV-2 genital herpes. Further, it is interesting to notethat polyethylene glycol dispersed acyclovir monophosphate showed thebest efficacy in treating lesions.

EXAMPLE XII Activity Of Acyclovir Diphosphate

The procedure of Example 8 is repeated using a formulation having onlyacyclovir diphosphate (ACV-DP) as the acyclovir derivative. Efficacysuperior to that of ACV alone is observed.

EXAMPLE XIII Activity Of Acyclovir Monophosphate Glycerol

The procedure of Example 8 is repeated using a formulation having onlyacyclovir monophosphate glycerol (ACV-MP-G) as the acyclovir derivative.Efficacy superior to that of ACV alone is observed.

EXAMPLE XIV Activity Of Acyclovir Diphosphate Glycerol

The procedure of Example 8 is repeated using a formulation having onlyacyclovir diphosphate glycerol (ACV-DP-glycerol) as the acyclovirderivative. Efficacy superior to that of ACV alone is observed.

EXAMPLE XV Activity Of Acyclovir Monophosphate Morpholidate

The procedure of Example 8 is repeated using a formulation having onlyacyclovir monophosphate morpholidate (ACV-MP-morpholidate) as theacyclovir derivative. Efficacy superior to that of ACV alone isobserved.

EXAMPLE XVI Activity Of Acyclovir Monophosphate Isopropylidene Glycerol

The procedure of Example 8 is repeated using a formulation having onlyacyclovir monophosphate isopropylidene glycerol (ACV-MP-isoP-G) as theacyclovir derivative. Efficacy superior to that of ACV alone isobserved.

EXAMPLE XVII Activity Of Acyclovir Diphosphate Isopropylidene Glycerol

The procedure of Example 8 is repeated using a formulation having onlyacyclovir diphosphate isopropylidene glycerol (ACV-DP-isoP-G) as theacyclovir derivative. Efficacy superior to that of ACV alone isobserved.

EXAMPLE XVIII Solubility Of Acyclovir Monophosphate And Salts

Various salts of acyclovir monophosphate were tested for solubility asfollows:

Two ml of distilled water was placed in each of three 10 ml beakers,each beaker having magnetic stirring bars. In each individual flask, anacyclovir monophosphate salt, selected from potassium, sodium,sodium/ammonium, and H⁺ (free acid), was added until a saturatedsolution was formed. Each saturated salt solution was gravity filtered.The acyclovir monophosphate sodium/ammonium and free acid salt solutionswere filtered through Whatman No. 4 filter paper and gave clearsolutions. The potassium and sodium salt solutions were filtered throughWhatman No. 1 filter paper and each gave slightly opalescent solutions.

One ml of each of the saturated salt solutions were transferred bypipette to preweighed round bottom flasks, and the solutions wereallowed to dry. After all of the liquid had evaporated, the round bottomflasks were reweighed and the number of milligrams of acyclovirmonophosphate salt present per milliliter was easily found.

The following Table sets forth the solubility of the various saltsprepared as described above relative to acyclovir:

                  TABLE III                                                       ______________________________________                                                      Solubility as                                                                 Compared to                                                     Salt          Acyclovir                                                       ______________________________________                                        H.sup.+        21 X                                                           K.sup.+        85 X                                                           Na.sup.+ /NH.sub.4.sup.+                                                                    100 X                                                           Na.sup.+      108 X                                                           ______________________________________                                    

It will be appreciated in view of the results shown in Table III, thatthrough formation of a salt of the acyclovir monophosphate, solubilitycan be dramatically enhanced. It is expected that other nucleosidemonophosphates will exhibit similarly enhanced solubility. In thismanner, it is possible to formulate topical compositions containinglarge quantities of acyclovir monophosphate because of the enhancedsolubility of the salts.

There will be various modifications, improvements, and applications ofthe disclosed invention that will be apparent to those skilled in theart, and the present application is intended to cover such embodiments.Although the present invention has been described in the context ofcertain preferred embodiments, it is intended that the full scope of thedisclosure be measured only by reference to the following claims.

What is claimed is:
 1. A method for treating a herpes virus infectionthat is resistant to acyclovir, comprising topically applying aneffective amount of an cyclovir phosphate ester, a pharmaceuticallyacceptable acyclovir phosphate ester salt, or a mixture thereof, in apharmaceutical carrier suitable for topical use, to the herpes virusinfected cutaneous or mucosal tissues of a herpes virus infected animal.2. The method of claim 1 wherein the animal is a human being.
 3. Themethod of claim 1 or 2, wherein the phosphate ester is acyclovirmonophosphate.
 4. The method of claim 1 or 2, wherein the phosphateester is acyclovir diphosphate.
 5. The method of any one of claims 1, 3,4 or 2, wherein the acyclovir phosphate ester is in the form of apharmaceutically acceptable salt.
 6. The method of claim 5, wherein thepharmaceutically acceptable salt is selected from the group consistingof sodium, potassium, and ammonium salts.
 7. The method of claim 1wherein the pharmaceutical carrier comprises a vehicle selected from thegroup consisting of an aqueous cream and polyethylene glycol.
 8. Themethod of claim 1 or 2 wherein the acyclovir resistance of the virus isdue to an alteration or defect in the viral gene coding for thymidinekinase.
 9. The method of claim 1 or 2 wherein the cutaneous or mucosaltissues are infected by herpes simplex, type 1 virus.
 10. The method ofclaim 1 or 2 wherein the cutaneous or mucosal tissues are infected byherpes simplex, type 2 virus.
 11. The method of claim 1 or 2 wherein theherpes virus infected tissues are in the orofacial region of the animalbody.
 12. The method of claim 1 or 2 wherein the herpes virus infectedtissues are in the genital region of the animal body.
 13. A method fortreating a herpes virus infection in which the herpes virus hasdeveloped a resistance to one or more antiviral compounds due to analteration or defect in the viral gene coding for thymidine kinase,comprising applying an effective amount of an acyclovir phosphate ester,a pharmaceutically acceptable acyclovir phosphate ester salt, or amixture thereof, in a pharmaceutical carrier suitable for topical use,to the herpes virus infected cutaneous or mucosal tissues of an animal.14. The method of claim 13 wherein the animal is a human being.
 15. Themethod of claim 13 or 14, wherein the phosphate ester is acyclovirmonophosphate.
 16. The method of claim 13 or 14, wherein the phosphateester is acyclovir diphosphate.
 17. The method of claim 13 or 14,wherein the phosphate ester is in the form of a pharmaceuticallyacceptable salt.
 18. The method of claim 17, wherein thepharmaceutically acceptable salt is selected from the group consistingof sodium, potassium, and ammonium salts.
 19. The method of claim 13 or14 wherein the pharmaceutical carrier comprises a vehicle selected fromthe group consisting of an aqueous cream and polyethylene glycol. 20.The method of claim 13 or 14 wherein the cutaneous or mucosal tissuesare infected by herpes simplex, type 1 virus.
 21. The method of claim 13or 14 wherein the cutaneous or mucosal tissues are infected by herpessimplex, type 2 virus.
 22. The method of claim 13 or 14 wherein theherpes virus infected tissues are in the orofacial region of the animalbody.
 23. The method of claim 13 or 14 wherein the herpes virus infectedtissues are in the genital region of the animal body.